Method of treating colorectal carcinoma

ABSTRACT

A method is disclosed for detecting colorectal carcinoma having at least one HERG potassium ion channel, where at least one HERG potassium channel is detected in a tissue biopsy of the human color or rectum. In addition, a method is disclosed for treating colorectal carcinoma having at least one HERG potassium channel in a patient in need of said treatment, which comprises the step of administering to said patient, a therapeutically effective amount of 4-[1-{2-(6-methyl-2-pyridinyl)ethyl-4-piperidinyl}carbonyl]methane-sulfoanilide 2HCl sufficient to treat the colorectal carcinoma having at least one HERG potassium channel by selectively blockading the HERG potassium channel.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of application Ser. No. 10/516,521filed 2 May 2005, now U.S. Pat. No. 7,598,270, which is the US NationalPhase of PCT/EP03/05710 filed 30 May 2003, which itself claims thepriority of German Application 102 24 534.7 filed 31 May 2002.

FIELD OF THE INVENTION

This invention relates to a diagnostic agent and a method for detectionof cancer, in particular a method for detection of colorectal cancer(cancer of the large intestine) in a tissue biopsy of the human colon orrectum and a means for treatment of carcinoma, in particular colorectalcarcinoma.

BACKGROUND OF THE INVENTION

Cancer is known to have become increasingly important in all countries.Treatment of cancer and therapeutic success depend to a significantextent on prompt detection of the cancer. Therefore, there is a greatdemand for reliable cancer diagnostic agents, in particular those thatallow detection of metastases and micrometastases, even when there areno definite histological findings or when the histological findings arenegative.

Colorectal cancer is the second main cause of cancer fatalities; theincidence has been increasing steadily and it often recurs after acurative surgical operation. Colorectal cancer (malignant tumors of thecolon and rectum) are occurring in a constantly increasing incidence inindustrial countries and constitute the second most common type ofcancer in men and the third most common in women. Colorectal cancerconstitutes 50% of malignancies. There are more than 200,000 new casesof colorectal cancer each year, and more than 100,000 patients die ofit. Colorectal cancer is thus the second leading cause of death due tocancer.

Colorectal cancer may develop de novo or as part of an adenoma-carcinomasequence in an adenomatous polyp. The probability of a malignancy isbetween 1% and 40% in the case of adenomas. To this extent, the patientswith colorectal polyps constitute a risk group.

For this reason, early detection of colorectal cancer in adenomas andreliable differentiation from benign colorectal tissue are of crucialimportance, especially for the prognosis and course of treatment.

Diagnosis and prognosis for this type of cancer are influenced by avariety of properties which are present at the time of the initialdiagnosis. These factors include age, sex, duration of symptoms,condition of the intestinal obstruction, tumor localization, the needfor a blood transfusion and the quality of the surgical intervention.Although a number of tumor properties such as vascular lymphaticinvasiveness, the degree of differentiation and the preoperative titerof conventional tumor markers have shown a prognostic relevance, butthere are no suitable markers for detection of early stages of cancer(benign colorectal precursors (adenomas) that become malignant) or forhistopathologically unremarkable micrometastases (minimal residualdisease) which may be responsible for a recurrence of the carcinoma,even after curative surgical resection. The tumor markers CEA, CK 19 andCK 20 which have been used in the past are indicative of the currentprognosis but are unreliable in a differential diagnosis.

OBJECT OF THE INVENTION

Therefore, the object of this invention is to make available a reliablediagnostic agent and a method for detection of colorectal cancer andalso to permit the use of an effective agent for treatment of colorectalcancer.

SUMMARY OF THE INVENTION

To achieve this object, this invention proposes that a tissue biopsy ofthe human colon or rectum should be tested for the presence of HERGpotassium channels.

Therefore, the object of this invention is a diagnostic agent fordetection of colorectal cancer with which the presence of at least oneHERG potassium channel in a tissue specimen of the human colon orrectum, which is free of HERG potassium channels in a healthy person,can be detected. Another object of the present invention is a method fordiagnosing colorectal cancer, whereby the presence of at least one HERGpotassium channel is detected in a tissue biopsy of the human colon orrectum, which is free of HERG potassium channels in a healthy person, orin lymph nodes or in a body fluid.

Detection of a HERG potassium channel with the diagnostic agentaccording to the present invention is of course a reliable indication ofthe presence of colorectal cancer only if the detection is performed ina tissue biopsy of the human colon or rectum or in lymph nodes or in abody fluid which is free of HERG potassium channels in a healthy person.This does not apply to myocardial tissue or the brain, where HERGpotassium channels always occur in a healthy person. However, if a HERGpotassium channel is detected in a tissue biopsy of the colon or rectum,for example, which are known not to normally have HERG potassiumchannels, then this is a reliable detection of the presence of cancer.

Detection of cancer based on the occurrence of HERG potassium channelsis reliable even if this detection is performed in a body fluid such asblood, blood plasma, blood serum, urine, perspiration or lacrimal fluidor feces in which HERG potassium channels normally never occur undernormal conditions.

An example of the inventive method and diagnosing a cancer consists ofdiagnosing a colorectal cancer by detecting at least one HERG potassiumchannel in a tissue biopsy of the human colon.

For the diagnosis, a tissue biopsy of the colon and/or rectum is testedin the laboratory, with the HERG potassium channel being expressed incolorectal carcinoma cells which can be detected by the highly sensitiveRT-PCR method as well as by the immunohistochemical tests that arewidely used in clinical practice.

The surprising and valuable finding is that the HERG potassium channelis a highly selective functional tumor marker for colorectal carcinomaand thus can be used in diagnostics, especially for detection of earlystages of cancer, for detection of unremarkable micrometastases and fordifferentiation from healthy colorectal tissue.

Members of the ether a-go-go (EAG) potassium channel family, inparticular the eag-dependent gene product (eag-related gene; ERG) suchas the human ERG (HERG) potassium channel are suitable for diagnosticpurposes. They can be found in various tumor cell lines of varyinghistogenesis; it is known that they play a role in cell proliferationand transformation.

The reverse transcriptase polymerase chain reaction (RT-PCR) is a veryefficient and highly sensitive method of detecting minimaltumor-associated mRNA transcription. Immunohistochemistry is a customaryand widespread method in clinical practice and is therefore easy tointegrate into routine techniques.

The HERG potassium channel (HERG=human eag-related gene) is a specialtype of human potassium channel which belongs to the eag (ether a-go-go)family of voltage-activated potassium channels. The HERG potassiumchannel evidently plays an important pathophysiological role in theregulatory mechanisms in neoplastic cells of varying histopathogenesisand triggers unlimited tumor growth.

The present invention proposes the use of the antiarrhythmic agentE-4031 for treatment of colorectal carcinoma cells. This is based on thevaluable finding that the functional HERG potassium channel that playsan important role in cell proliferation and in carcinoma growth can beblocked selectively by the antiarrhythmic agent E-4031. This allows newapproaches in cancer therapy.

Thus the invention is directed to a method of treating a colorectalcarcinoma having at least one voltage-activated potassium ion channelHERG subunit in a patient in need of said treatment, which comprises thestep of administering to said patient a therapeutically effective amountof4-[1-{2-(6-methyl-2-pyridinyl)ethyl-4-piperidinyl}carbonyl]methane-sulfoanilide2HCl sufficient to treat the colorectal carcinoma having at least onevoltage-activated potassium ion channel HERG subunit. In particular theinvention is directed to a method of treating colorectal carcinomahaving at least one voltage-activated potassium ion channel HERG subunitin a patient in need of said treatment, which comprises the step ofadministering to said patient, a therapeutically effective amount of4-[1-{2-(6-methyl-2-pyridinyl)ethyl-4-piperidinyl}carbonyl]methane-sulfoanilide2HCl sufficient to treat the colorectal carcinoma having at least onevoltage-activated potassium ion channel HERG subunit by selectivelyblockading the HERG subunit.

The antiarrhythmic agent E-4031 is a4-[1-[2-(6-methyl-2-pyridinyl)ethyl-4-piperidinyl]carbonyl]methanesulfoanilide.2HCl of the following formula:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a showing RT/PCR amplification for detecting CEA, CK-19, CK-20and HERG mRNA expression in healthy colon tissue, in tubulovillousadenocarcinomas of the colon, and in colorectal carcinoma.

FIG. 2 is a series of 4 graphs depicting the inhibition of proliferationof the Colo-205 colorectal carcinoma cell line, at an initialinoculation of 10,000 cells per well by the selective inhibitor E-4031of the HERG potassium channel, employing Compound E-4031 at 0 μM(control), 1 μM, 5 μM and 10 μM concentrations, respectively.

FIGS. 3 and 4 show a staining pattern indicating animmunohistochemically positive reaction of the anti-ERG1 (HERG) antibodyin colorectal cancer.

FIG. 5 shows an immunohistochemically negative reaction in normalhealthy colorectal tissue.

FIG. 6 shows a weak immunohistochemically positive reaction for basalepithelial cells in the histopathologically unremarkable remainingintestinal specimen of the resected tissue of a tumor patient with laterlocoregional recurrence of the cancer.

Tests were performed on 24 tissue biopsies from 18 different patients(twelve women and six men between the ages of 51 and 86, average age67.7 years) with colorectal cancer of UICC classifications I through IV.Five cancers were located in the colon, four were located in the sigmoidcolon and nine were located in the rectum. The tissue biopsies wereexcised from the surrounding tissue to prevent cross-contamination. Anew scalpel was used for each individual tissue biopsy.

The clinical and histopathological UICC classification as well as theDukes' classification of patients with colorectal adenocarcinoma yieldedthe following result:

Stage I—Duke A: 3 (pT1=1, pT2=2)

Stage III—Duke B: 5 (pT3=5, pT4=0)

Stage III—Duke C: =7 (pN1=4, pN2=3)

Stage IV—Duke D: 3 (pM1=3)

Histopathologically negative biopsies of colon tissue of seven patients(four women and three men between the ages of 60 and 68, average age62.8), three of which were from a tubulovillous adenoma of the colon andfour of which were from a sigmoid diverticulitis were used as thenegative controls (Table 2).

The histopathological diagnoses, UICC classification and Dukes'classification as well as the TNM classification were performed understandardized conditions. The tissue biopsy was deep-frozen in liquidnitrogen immediately after excision and stored at −80° C. until RNAextraction.

All the cellular RNA isolation from the frozen tissue biopsy wasperformed using the Qiagen RNeasy mini-kit according to themanufacturer's instructions (Hilden, Germany). The amount and purity ofthe RNA were determined by spectrophotometry at wavelengths of 260 nmand 280 nm.

The Colo-205 colorectal carcinoma cell line was used as a positivecontrol for detecting the sensitivity of these experiments.

Reverse Transcription

The cDNA was synthesized from 2 μg total RNA in a volume of 20 μLreaction mixture containing 4 μL of 5× reaction buffer (50 mmol/LTris-HCI, pH 8.3, 75 mmol/L KCl and 3 mmol/L MgCl₂), 500 μmol/L DNTP,100 μrnol/L solution of poly-dT15 primer (Roche Diagnostic, Mannheim,Germany) and 500 units of Superscript II (Gibco BRL, Gaithersburg, Md.,USA). The mixture was incubated for 60 minutes at 42° C., then heatedfor two minutes at 90° C. and next cooled on ice.

Primer sequences for the RT-PCR analysis:

The following primer sequences were used for the subsequent RT-PCRtests:

The primer sequences for CEA mRNA were:

A. 5′-TCTGGAACTTCTCCTGGTTCTCTCAGCTGG-3′ (SEQ ID NO:1) for the outersense;

B. 5′-TGTAGCTGTTGCAAATGCTTTAAGGAAGAA-3′ (SEQ ID NO:2) for the antisense;and

C. 5′-GGGCCACTGTCGGCATCATGATTGG-3′ (SEQ ID NO:3) for the inner sensecases.

The primer sequences for CK-19 mRNA were:

A. 5′-GTGGAGGTGGATTCCGCTCC-3′ (SEQ ID NO:4) for the outer sense;

B. 5′-TGGCAATCTCCTGCTCCAG-3′ (SEQ ID NO:5) for the outer antisense;

C. 5′-ATGGCCGAGCAGAACCGGAA-3′ (SEQ ID NO:6) for the inner sense; and

D. 5′-CCATGAGCCGCTGGTACTCC-3′ (SEQ ID NO: 7) for the inner antisensecases.

The primer sequences for CK-20 mRNA were:

A. 5′-GCGTTTATGGGGGTGCTGGAG-3′ (SEQ ID NO:8) for the outer sense;

B. 5′-AAGGCTCTGGGAGGTGCGTCTC-3′ (SEQ ID NO:9) for the outer antisense;

C. 5′-CGGCGGGGACCTGTTTGT-3′ (SEQ ID NO: 10) for the inner sense; and

D. 5′-CAGTGTTGCCCAGATGCTTGTG-3′ (SEQ ID NO: 11) for the inner antisensecases.

The primer sequences for the HERG mRNA were:

A. primer up 5′-AGCTGATCGGGCTGCTGAAGACTG-3′ (SEQ ID NO:12) and

B. primer down 5′-AATGAGCATGACGCAGATGGAGAAG-3′ (SEQ ID NO:13).

To investigate the integrity of the extracted RNA and to ensure thatequimolar RNA was used, the extracted RNA was tested withglyceraldehyde-3-phosphate dehydrogenase (GAPDH) by RT-PCR. The primarysequences for GAPDH were:

5′-CCACCCATGGCAAATTCCATGGCA-3′ (SEQ ID NO:14) sense and

5′-TCTAGACGGCAGGTCAGGTCCACC-3′ (SEQ ID NO:15) antisense primers.

Reverse transcriptase polymerase chain reaction (RT•PCR):

Two-step RT-PCR was used for amplification of cDNA of CEA, CK-19 andCK-20 as well as HERG.

PCR was performed as follows: PCR was performed in a volume of 50 μL and2 μg of the total RNA per sample. For the first PCR round, 2 μL,aliquots of the cDNA solution were mixed with 10.5 μL of the PCRreaction mixture containing 1.25 μL 10×PCR buffer (10 mmol/L Tris-HCl,pH 8.3, 50 mmol/L KCl and 1.5 mmol/l MgCl₂), 200 μmol/L DNTP, 0.5 μmol/Lof each primer and 2.5 units of platinum Taq polymerase (Gibco BRL,Gaithersburg, Md.).

The reaction was continued in a PCR Thermocycler (Biometra, Gottinqen,Germany). For CEA, CK-19 and CK-20 amplification, the followingconditions were used: activation of Taq polymerase for four minutes at95° C., template denaturing for 45 seconds at 95° C., annealing for 45seconds at 60° C. and elongation for 45 seconds at 72° C. for 20 cycles.HERG amplification was performed for four minutes at 95° C., for oneminute at 55° C., for one minute at 72° C. and for one minute at 95° C.for 30 cycles. Two microliters of the first PCR product were transferredto a second tube and amplified further (CEA, CK-19 and CK-20: for fourminutes at 95° C.; 45 sec at 95° C.; 45 sec at 60° C. and 45 sec at 72°C. in 20 cycles and HERG for four minutes at 95° C.; one minute at 55°C.; one minute at 72° C. and one minute at 95° C. in 30 cycles). Allpipetting work was performed on ice while working at a sterile workbenchand then the tubes were placed in the Thermocycler. First however, theywere heated to the denaturing temperature before amplification wasstarted.

The amplified DNA fragments were 132 base pairs (bp) for CEA primerpairs, 328 base pairs for CK-19 primer pairs, 485 base pairs for CD-20primer pairs and 386 base pairs for HERG primer pairs. Each tissuebiopsy was tested at least twice.

To check on the integrity for cDNA, amplification for GAPDH wasperformed in 25 cycles with 603 base pairs of the cDNA fragments (fourminutes at 95° C.; 45 sec at 95° C.; 45 sec at 60° C.; and 45 sec at 72°C.).

The PCR products were applied to a 2% agarose gel and stained withethidium bromide.

Sensitivity Tests of the RT-PCR Method

The Colo-205 colorectal cancer cell line was used as a positive controlfor the detection sensitivity in these experiments. To determine thesensitivity of the method, dilution series of 10⁶ to 10⁰ colorectalcarcinoma cells (CoI0-205) were prepared and then 10⁷ lymphocytes fromhealthy donors were added. RT-PCR was performed after extraction of thetotal RNA. The dilution series of the RNA showed that the primers werecapable of detecting a quantity of mRNA equivalent to one tumor cell in10⁶ lymphocytes.

Immunohistochemistry

Biopsies of colorectal carcinomas and healthy colorectal tissue werefixed in buffered formalin and embedded in paraffin. The histology wasevaluated on sections stained with hematoxylin and eosin. Additionalsections were used for the immunohistochemical verification.

For the immunohistochemistry of the anti-HERG antibody, a protocol ofthe ABC (avidin-biotin-peroxidase complex) detection method was used.

After applying the sections to microscope slides and air drying themovernight at 37° C., deparaffinization was performed with xylene andrehydration was performed with a descending alcohol series. Theendogenous peroxidase was blocked by immersion of the sections for tenminutes in a solution of 19 mL phosphate buffer, 1 mL methanol and 200μL 30% H₂O₂. Then the sections were rinsed with PBS 6×10 min.

To reduce the nonspecific antibody binding, the sections werepreincubated with normal goat serum.

Then the sections were incubated for one hour at 4° C. with rabbitanti-ERG1 (HERG) (AB5222-200UL, Chemicon, Temecula, Calif., USA) as theprimary antibody in a 1:500 dilution. After rinsing three times withPBS, the sections were incubated in biotinylated anti-rabbit antibody(Vector) in a dilution of 1:200 for one hour at 25° C. Then the sectionswere rinsed three times with PBS. Next they were incubated with the ABCcomplex (Vector) for one hour at 25° C. After rinsing several times, thesections were treated with 3,3′-diaminobenzidine tetrahydrochloride(DAB) (Sigma) for three minutes at 25° C. according to a standardprotocol (0.05% DAB in 0.1 M phosphate buffer and 0.3% H₂O₂) forvisualization of the primary antibody.

The staining of the sections was compared under a microscope. Thestaining reaction was stopped by rinsing with PBS.

When a positive antibody reaction was obtained, the affected tissue areawas stained brown homogeneously.

After immunohistochemistry, the sections were dried overnight,dehydrated and then covered.

Negative controls: same procedure without primary antibody.

Positive controls: experiments with anti-ERG1 (HERG) antibody wereperformed on sections of human heart.

Proliferation test on the Colo-205 colorectal carcinoma cell line withE-4031

Proliferation tests (growth experiments) were performed with the3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide MTTtest. Cells of the Colo-205 colorectal carcinoma cell line wereharvested with 0.05% EDTA and then counted. The cells were then sown ina 96-well plate with 10⁴ live cells per well and left for 24 hours toadhere. After 24 hours, the medium was exchanged with E-4031 in variousconcentrations of 1 μm, 5 μM and 10 μM. The final volume was 200 μL perwell. After incubating for 0 to 7 days, 20 μL MTT (5 mg/mL in PBS) wasadded and incubated for two hours at 37° C. The medium was then removedand 100 dimethyl sulfoxide (Sigma, Germany) was placed in each well forten minutes.

A 96-well microtiter plate reader (Comtek, Germany) was used for theanalysis. At a wavelength of 570 nm angstrom the MTT test was read inthe ELLSA microtiter plate reader. The average concentration in a set offive wells was measured.

The absorption of the untreated controls was assumed to be 100% and theIC₅₀ was calculated accordingly.

Statistical Analysis

A difference of P<0.05 was assumed to be significant.

Results of RT-PCR

CEA, CK-19 and CK-20 were expressed in the carcinoma, in the controltissue and in the Colo-205 colorectal cell line. HERG expression,however, was 100% limited to only the carcinoma tissue and the Colo-205cells. As shown in FIG. 1 and Table 1, tissue biopsies which definitelyshow a colorectal carcinoma in the histopathological examination alsoshowed expression of all markers (CEA, CK-19, CK-20 and HERG).Colorectal tissue from patients without a malignancy would also expressCEA, CK-19 and/or CK-20, but no HERG could ever be detected (Table 2).When RT-PCR was performed without the RT enzyme, no bands could bedetected in the Colo-205 cell line, which proves that no DNA was presentand thus the RT-PCR conditions are adjusted very well.

In columns 1 and 15, FIG. 1 shows 100 base pair increments as controlbands. Column 2 shows the GAPDH control which was positive in allsamples. The results in columns 3 through 6 are from a tissue biopsy ofa patient with sigmoid diverticulitis (patient no. 6 in Table 2) and theresults in columns 7 through 10 were obtained on a tissue biopsy of apatient with a tubulovillous adenoma of the colon (patient no. 1 inTable 2). The bands in columns 11 through 14 represent a tissue biopsyof patient with a colorectal carcinoma (patient no. 3 in Table 1). TheDNA fragments are applied to a 2% agarose gel and stained with ethidiumbromide.

As summarized in Table 1, CEA can be detected in 22 (91.7%) tissuebiopsies of colorectal carcinomas (of 18 different patients tested),CK-19 can be detected in 19 tissue biopsies (79.2%), CK-20 in 23 tissuebiopsies (95.8%) and HERG alone can be detected in 100% i.e., all 24tissue biopsies.

In all samples, except for CK-19 and CD-20 expression in patient no. 2,there were no differences in the mRNA results of the various tissuebiopsies from the same test preparation (patients nos. 4, 6, 7, 8, 9)(Table 1).

TABLE 1 Detection of mRNA expression of tumor markers CEA, CK-19, CK-20and HERG by RT-PCR in colorectal carcinomas Patient GAPDH CEA CK-19CK-20 HERG Staging TNM Localization  1 pos pos pos pos pos III-T4, N1(2/10), MO Sigmoid  2a pos pos neg pos pos III-T3, N1 (1/9), MO Colon 2b pos pos pos pos pos  3 pos pos neg pos pos II-T3, NO (0.15), MOColon  4a pos pos pos pos pos IV-T3, N2 (25/27), M1 Rectum  4b pos pospos pos pos  5 pos neg pos pos pos III-T2, N1 (2/12), MO Sigmoid  6a pospos pos pos pos III-T3, N1 (1/35), MO Rectum  6b pos pos pos pos pos  7apos pos neg pos pos III-T3, N2 (21/27), MO Rectum  7b pos pos neg pospos  8a pos pos pos pos pos I-T2, NO (0/10), MO Colon  8b pos pos pospos pos  9a pos pos pos pos pos II-T3, NO (0.4), MO Rectum  9b pos pospos pos pos 10 pos pos pos pos pos IV-T3, N2 (6/16), M1 Rectum 11 pospos pos pos pos II-T3, NO (0/4), MO Colon 12 pos pos pos pos pos IV-T3,N2 (6/16), M1 Sigmoid 13 pos pos pos pos pos II-T3, NO (0/7), MO Rectum14 pos pos pos pos pos II-T3, NO (0/13), MO Colon 15 pos pos pos pos posIII-T3, N2 (6/11), MO Rectum 16 pos pos pos pos pos I-T1, NO (0/16), MOSigmoid 17 pos pos pos pos pos III-T3, N2 (6/36), MO Rectum 18 pos negneg pos pos II-T3, NO (0/16), MO Rectum

The grading (degree of histopathological differentiation) of thecolorectal carcinomas was G2 for all tissue biopsies except the biopsyfrom patient no. 1 (G2-3), patient no. 4 (G3) and patient no. 9 (G2-3).

HERG mRNA was not detected in any (0%) of the histopathologicallynegative colorectal tissue biopsies of three patients with atubulovillous adenoma of the colon and the four patients with sigmoiddiverticulitis who served as negative controls (FIG. 1 and Table 2).

The mRNA of the tumor markers CEA, CK-19 and CK-20 was positive in allpatients with an adenoma of the colon (100%). The mRNA of the markersCEA, CK-19 and CK-20 was positive in two patients (50%) in the tissuebiopsies of the patients with sigmoid diverticulitis.

TABLE 2 Detection of mRNA expression of tumor markers CEA, CK-19, CK-20and HERG by RT-PCR in histopathologically negative colorectal tissue.Patient GAPDH CEA CK-19 CK-20 HERG Histology Localization 1 pos pos pospos neg tubulovillous adenoma Colon 2 pos pos pos pos neg tubulovillousadenoma Colon 3 pos pos pos pos neg tubulovillous adenoma Colon 4 pospos neg neg neg sigmoid diverticulitis Sigmoid 5 pos pos pos pos negsigmoid diverticulitis Sigmoid 6 pos neg pos pos neg sigmoiddiverticulitis Sigmoid 7 pos neg neg neg neg sigmoid diverticulitisSigmoidResults of Immunohistochemistry

Results of immunohistochemical detection of the anti-ERG1 (HERG)antibody definitely show an immunohistochemically positive reaction bystaining of the malignant (glandular) tissue components in question inall biopsies of the colorectal carcinoma (Table 3). The immunereactivity is limited to the basal part of the cytoplasm of theepithelial cells (FIGS. 3 and 4). In addition to epithelial cells,diffuse reactions can also be detected in the lamina propria and in theblood vessels (blood cells). In addition, isolated cells notcharacterized further are found in the muscle layers.

The remaining intestinal sections of the resected material from the sametumor patient that were histopathologically negative were alsoimmunohistochemically negative in most tissue biopsies. However, inisolated cases of immunohistochemically positive tissue biopsies, a weakbut definitely positive immune reaction was found in the basalepithelial cells at the base of the glands in the remaining sections ofthe resected intestine that were histopathologically negative (FIG. 6).In a representative number of patients, a locoregional recurrence of thecarcinoma was detected within a period of two years after surgicalresection of the colorectal carcinoma (patients nos. 19-25 in Table 3).

This sensitive and reliable immunohistochemical detection of HERG inhistopathologically unremarkable tissue and thus the detection ofmicrometastases make HERG a reliable tumor marker.

In no case did the histopathologically negative colorectal tissuebiopsies of benign intestinal changes (sigmoid diverticulitis,colorectal adenomas) or healthy colorectal tissue biopsies (regularhealthy intestinal mucosa) show staining in any cases and thus they gavea negative immunohistochemical reaction (Table 4 and FIG. 5).

TABLE 3 Immunohistochemical results in the colorectal carcinoma tissueand in the histopathologically negative remaining colorectal tissue ofthe same tumor patient (of patients with continuous numbers 1 through25, patients nos. 1 through 18 are included in PCR). Remaining intestineof the resected tissue Patient Carcinoma tissue histologically negative 1-18 Positive Negative 19-25 Positive Positive

TABLE 4 Immunohistochemical results in histopathologically negativetissue biopsies (of the patients with continuous nos. 1 through 25,patients nos. 1 through 7 of PCR are included). Patient Regular healthyintestinal mucosa 1-6 Negative Sigmoid diverticulitis  7-18 NegativeColorectal adenoma 19-25 NegativeResults of the Proliferation Assay

FIG. 2 shows the inhibition of proliferation of the Colo-205 colorectalcarcinoma cell line by the selective inhibitor E-4031 of the HERGpotassium channel.

E-4031 was supplied to the cells in three different concentrations (1, 5and 10 μM) and then the growth response of the colorectal carcinomacells was measured on the five following days. Growth of the carcinomacells was inhibited by the concentrations of E-4031, due to the blockingof the HERG potassium channel, which is important for the growth ofcancer, and then the cancer cells died off.

Thus according to the present invention, a tumor marker has beendiscovered which makes it possible to reliably differentiate cancerous(malignant) tissue from non-cancerous (benign) colorectal tissue andthus to detect cancer and also detect cancerous tumor cells inhistopathologically unremarkable tissue. In colorectal adenomas, this isespecially important for reliable early detection of carcinoma cells andfor reliable detection of micrometastases in histopathologicallyunremarkable tissue, and in the lymph nodes it is important for thecourse of treatment and for the prognosis.

CEA is not suitable as such a marker because CEA mRNA has not beendetected in stage I colorectal carcinomas but it is expressed in alltissue biopsies of tubulovillous colon adenomas and in two samples ofsigmoid diverticulitis which functioned as negative controls. It isconsistent with the findings presented here that Bhatnagar et al. found(by means of quantitative enzyme immunoassay and immunocytochemistry)that CEA is not present at all in moderately to slightly differentiatedcolorectal tumors. Boucher et al. demonstrated that CEA occurs not onlyin adenocarcinoma of the colon but also in healthy adjacent mucosaltissue of the colon.

CK-19 and CK-20 occur not only in serum but only in colorectal tissue sothey are not reliable tumor markers. They cannot be detected in advancedstage III carcinoma, but they can be detected in some control tissuebiopsies. This confirms previous observations that CK-19 and CK-20 canalso be discovered in healthy intestinal mucosa but are not present insome tumors. The observation that the abundant occurrence of CK-19 inwell-differentiated tumor tissue and in low-grade dysplasia with slightcellular proliferation as well as the low occurrence in slightlydifferentiated carcinoma tissue and in high-grade dysplasia with a highproliferation rate as well as the slight expression of CK-20 incolorectal carcinoma in comparison with healthy colon tissue all suggestthat the negative tumor biopsies of stage III is in a high proliferationstatus. The contrary findings of the tissue biopsy from patient no. 2(Table 1) additionally show that the markers are not distributeduniformly in the tumor.

In contrast with the findings for CEA, CK-19 and CK-20, HERG expressionwas found selectively in all colorectal carcinomas regardless of thetumor stage and was negative in all control tissue biopsies. This can bedetected on a molecular biological level by detection of HERG RNA usingthe PCR method as well as by immunohistochemistry by detection of theHERG protein selectively in the colorectal carcinoma cells.

HERG-positive reactions were detected by immunohistochemical methods insome tumor patients In some histopathologically negative residualintestinal components of resected tissue from tumor patients. In the twoyears following surgical resection of the carcinoma, these patientsexperienced a locoregional recurrence of the colorectal carcinoma. Thisis a sign of micrometastases in histopathologically unremarkablecolorectal tissue.

By detection of HERG with two sensitive independent methods, both ofwhich are conventional, there is an enormous gain in the quality andreliability of detection results.

Therefore, the HERG potassium channel is a highly selective tumor markerfor colorectal tissue.

It is also a valuable marker for other epithelial carcinomas because italso occurs in endometrial cancer. The data presented suggests a stronginvolvement of the EAG potassium channels, including the HERG potassiumchannel, which has recently been suspected on the basis of in vitrostudies, in the carcinogenesis. These potassium channels have been foundin various tumor cell lines which transfer a transformed phenotype intotransfected cells of mammals and favor tumor progression inimmunosuppressed mice. Since the HERG potassium channel is of immenseimportant for tumor growth and/or survival of the tumor, proliferationstudies have shown that by selective blockade of the HERG potassiumchannel by E-4031, the carcinoma cells are induced to die off. The HERGpotassium channel as a functional tumor marker for colorectal carcinomais thus not only a selective tumor marker but also permits a newapproach for cancer therapy through selective inhibition of E-4031.

The surprising and valuable findings of this invention can be summarizedby stating that a reliable diagnosis of colorectal carcinoma cells ispossible by selective HERG gene expression in two independent testmethods (immunohistochemistry and PCR) with a novel and highly selectivemarker being used in the form of the HERG potassium channel, and thegrowth of tumor cells being inhibited by the antiarrhythmic agentE-4031.

1. A method of treating a colorectal carcinoma having at least onevoltage-activated potassium ion channel HERG subunit in a patient inneed of said treatment, which comprises the step of administering tosaid patient a therapeutically effective amount of4-[1-{2-(6-methyl-2-pyridinyl)ethyl-4-piperidinyl}carbonyl]methane-sulfoanilide2HCl sufficient to treat the colorectal carcinoma having at least onevoltage-activated potassium ion channel HERG subunit.
 2. A method oftreating colorectal carcinoma having at least one voltage-activatedpotassium ion channel HERG subunit in a patient in need of saidtreatment, which comprises the step of administering to said patient, atherapeutically effective amount of4-[1-{2-(6-methyl-2-pyridinyl)ethyl-4-piperidinyl}carbonyl]methane-sulfoanilide2HCl sufficient to treat the colorectal carcinoma having at least onevoltage-activated potassium ion channel HERG subunit by selectivelyblockading the HERG subunit.
 3. A method of treating colorectalcarcinoma having at least one voltage-activated potassium ion channelHERG subunit in a patient defined in claim 2 wherein the colorectralcarcinoma having at least one voltage-activated potassium ion channelHERG subunit is located in the colon, the sigmoid colon or the rectum.